Molecular cloning of a DNA-end-binding protein (Ku antigen) recognized by autoantibodies and its application.

PSS-PM重叠综合征患者的抗ku自身抗体识别与dsDNA末端结合的70kD/80kD蛋白异二聚体。在这个项目中，我们分离和鉴定了编码Ku自身抗原的cDNA克隆，并打算将其应用于临床。第一年，用抗Ku血清筛选插入到lambda gt11噬菌体EcoR1位点的人肝癌细胞cDNA文库，以鉴定表达Ku表位的斑块。分离和鉴定了3个阳性克隆(K14、K68和K71)，以表达抗Ku抗体识别的融合蛋白。第二年，用先前分离的编码80kD部分序列(K71)的cDNA作为探针进行菌落杂交，筛选出Okayama-berg文库。从K71与K71杂交的两个mRNAs中分离到含有最大插入片段(3.4kb)的克隆Ku80-6，并用Sanger‘s双脱氧法测定了其核苷酸序列。Ku80-6cDNA含有一个…编码732个氨基酸(MR=82,713)的单一长开放阅读框，其次是1082个碱基的3‘-非编码区和一个Poly(A)尾巴。使用计算机搜索将Ku80-6的序列与先前描述的序列进行比较，但在DNA(GenBank)或蛋白质(NBRF)数据库中都没有发现显著的同源性。在Southern杂交中，Ku80-6与人白细胞DNA经各种限制性内切酶消化后的4-5条DNA条带杂交。注意到RFLP出现在3.4kb-HindIII片段中，可能与SLE患者有抗ULRNP抗体有关。用纯化的K68(70kD)和K71(80kD)克隆表达的融合蛋白，建立了检测抗Ku抗体的酶联免疫吸附试验。用该方法筛选各种胶原病患者的血清时，抗Ku抗体主要在重叠综合征患者中检出，这与常规免疫扩散法的结果一致，我们的Ku抗原蛋白编码基因将为研究Ku自身抗原的结构和功能以及自身免疫性疾病的发病机制提供有力的工具。较少

Anti-ku autoantibodies in patients with PSS-PM overlap syndrome recognize a 70kD/80kD protein heterodimer which binds to ends of dsDNA. In this project, we isolated and characterized cDNA clones that encode the Ku autoantigen, and intended their clinical application.In the first year, a human hepatoma cell cDNA library inserted into the EcoRl site of lambda gtll phages were screened with an anti-Ku serum to identify plaques expressing Ku epitopes. Three positive clones (K14, K68 and K71) were isolated and deomnstrated to express fusion proteins recognized by anti-Ku antibodies. In the second year, Okayama-berg cDNA library was screened by using a previously isolated cDNA encoding the partial 80kD sequence (K71) as a probe for colony hybridization. The clone Ku80-6 that contained the longest cDNA insert (3.4kb, ldentical with the larger mRNA from two mRNAs hybridized by K71) was isolated, and its nucleotide sequence was determined by Sanger's dideoxy method. The Ku80-6 cDNA contained a … More single long open reading frame encoding 732 amino acids (Mr=82,713) followed by 1082 bases of 3'-non-coding region and a poly(A) tail. The sequence of the Ku80-6 was compared with previously described sequences using the computer search, but no significant homology was found either in DNA (GenBank) or protein (NBRF) data bank. In Southern blot, the Ku80-6 hybridized with 4-5 DNA bands from human leukocyte DNA digested with various restriction enzymes. It was noted that RFLP was seen in the 3.4kb-HindIII fragment and likely to associate with SLE patients who had anti-ULRNP antibodies. Using purified fusion proteins expressing from K68(70kD) and K71(80kD) clones, we developed ELISA to detect anti-Ku antibodies. When sera from various collagen diseases were screened by this assay, anti-Ku antibodies were mostly detected in patients with overlap syndrome, consisted with the result of a conventional immunodiffusion assay.Our cDNA encoding the Ku antigenic proteins will be a powerful tool to study not only the structure and function of the Ku antoantigen but also pathogenetic mechanisms of autoimmune diseases. Less